Use of recombinant gene delivery vectors for treating or preventing lysosomal storage disorders

ABSTRACT

Gene delivery vectors, such as, for example, recombinant FIV vectors, and methods of using such vectors are provided for use in treating or preventing retinal diseases of the eye and diseases of the brain associated with lysosomal storage disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of patent application Ser. No.09/580,820, filed May 26, 2000 now U.S. Pat. No. 6,730,297, from whichpriority is claimed under 35 USC § 120, and is related to provisionalpatent application Ser. No. 60/136,527, filed May 28, 1999 from whichpriority is claimed under 35 USC § 119(e)(1) and which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to compositions and methods for treatingdiseases of the eye, and more specifically, to the use of various genedelivery vectors which direct the expression of selected gene productssuitable for treating or preventing diseases of the eye and brainassociated with lysosomal storage disorders and other genetic defects.

BACKGROUND OF THE INVENTION

Mucopolysaccharidoses (MPS) refer to a group of inherited lysosomalstorage diseases, each of which is caused by the deficiency of alysosomal enzyme that degrades glycosaminoglycans (GAGs). MPS patientsexhibit undegraded GAGs in lysosomes, leading to lysosomal distentionand progressive cellular and organ dysfunction, caused by accumulationof chondroitin, dermatan and heparan sulphate. Patients afflicted withMPS can have a variety of clinical features including short stature,progressive bone and joint abnormalities termed dysostosis multiplex,course facial features, deafness, corneal clouding, hepatosplenomegaly,mental retardation and premature death. The lysosomal storage defect canoccur in the viscera, brain and skeleton, and the accumulated GAGs havea fibrillogranular appearance ultrastructurally (Vogler et al., J.Inher. Metab. Dis. 21:575–586, 1998).

One member of this disease group is a hereditary retinal disease causedby β-glucuronidase deficiency. Also known as MPS VII, it is aprogressive condition, with most tissues affected including the CNS.

Canine and murine models of MPS VII have been described (Haskins et al.,Pediatr Res 18:980–984, 1984, Birkenmeier et al., J Clin Invest83:1258–1256, 1989). The MPS mouse shares many common features withhuman patients, including the ocular pathology (Li and Davidson, PNAS92:7700–7704, 1995; Volger et al., Am J Pathol 136:207–217, 1990). Theseshared features make the MPS mouse an attractive model for studyingexperimental treatment of a lysosomal disease. For example, cells indiseased tissues contain numerous distended lysosomes. In the brain,both neurons and cells of glial lineage are affected. In the eye, theretinal pigment epithelium (RPE) is affected.

Gene therapy has been used to treat a variety of disorders and genetransfer to the eye has been attempted using recombinant vectors such asadenovirus (Li et al., Invest Opthalmol Vis Sci 35:2543–2549, 1994;Borras et al., Gene Ther 6:515–524, 1999; Li and Davidson, PNAS92:7700–7704, 1995; Sakamoto et al., H Gene Ther 5:1088–1097, 1999)adeno-associated virus (Ali et al., Hum Gene Ther 9:81–86, 1998,Flannery et al., PNAS 94:6916–6921, 1997; Bennett et al., InvestOpthalmol Vis Sci 38:2857–2863, 1997; Jomary et al., Gene Ther4:683–690, 1997, Rolling et al., Hum Gene Ther 10:641–648, 1999; Ali etal., Hum Mol Genet 5:591–594, 1996) and human immunodeficiency virus(Miyoshi et al., PNAS 94:10319–23, 1997; Takahashi et al., J Virol73:7812–7816, 1999). Each of these viruses infect slightly differentpopulations of cells. For example, an intravitreal injection ofadenovirus infects cells only in the anterior segment of the eye, mainlythe corneal endothelium and iris pigmented epithelium, while asubretinal injection results mainly in positive RPE and muller cells (Liet al., Invest Opthalmol Vis Sci 35:2543–2549, 1994; Li and Davidson,PNAS 92:7700–7704, 1995; Sakamoto et al., H Gene Ther 5:1088–1097, 1999.AAV injected intravitreally results in transduction of the ganglion-celllayer and the RPE. A subretinal injection produces positivephotoreceptors, in addition to the RPE and ganglion cells (Ali et al.,Hum Mol Genet 5:591–594, 1996). Studies with HIV injected subretinallyhave shown efficient transduction of the RPE and photoreceptors (Miyoshiet al., PNAS 94:10319–23, 1997; Takahashi et al., J Virol 73:7812–7816,1999).

Recombinant retroviral gene delivery methods have been extensivelyutilized in other gene therapy approaches, in part due to: (1) theefficient entry of genetic material (the vector genome) into cells; (2)an active, efficient process of entry into the target cell nucleus; (3)relatively high levels of gene expression; (4) the potential to targetparticular cellular subtypes through control of the vector-target cellbinding and the tissue-specific control of gene expression; (5) ageneral lack of pre-existing host immunity; (6) substantial knowledgeand clinical experience which has been gained with such vectors; and (7)the capacity for stable and long-term expression.

Briefly, retroviruses are diploid positive-strand RNA viruses thatreplicate through an integrated DNA intermediate. Upon infection by theRNA virus, the retroviral genome is reverse-transcribed into DNA by avirally encoded reverse transcriptase that is carried as a protein ineach retrovirus. The viral DNA is then integrated pseudo-randomly intothe host cell genome of the infected cell, forming a “provirus” which isinherited by daughter cells.

One type of retrovirus, the murine leukemia virus, or “MLV”, has beenwidely utilized for gene therapy applications (see generally Mann et al.Cell 33:153, 1983; Cane and Mulligan, PNAS 81:6349, 1984; and Miller etal., Human Gene Therapy 1 :5–14, 1990). One major disadvantage ofMLV-based vectors, however, is that the host range (i.e., cells infectedwith the vector) is limited, and the frequency of transduction ofnon-replicating cells is generally low.

Feline immunodeficiency virus (“FIV”)-mediated gene therapy vectorsystems have also been described (see, International Publication Nos. WO99/15641 and WO 99/36511).

The present invention provides compositions and methods for treating andpreventing a number of retinal and brain diseases and degenerations suchas RP and AMD, using retrovirus-mediated gene transfer and, further,provides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treating,preventing, or inhibiting diseases of the eye and the brain, and inparticular, diseases of the eye and brain that result from lysosomalstorage disease or from deficiency of retinal gene products. Within oneaspect of the present invention, methods are provided for treating orpreventing diseases of the eye or brain comprising the step ofintravitreal administration of a gene delivery vector which directs theexpression of one or more polypeptides, proteins or enzymes, such thatthe disease is treated or prevented. Within certain embodiments of theinvention, a viral promoter (e.g., CMV), a tissue-specific promoter(e.g., opsin or RPE), or an inducible promoter (e.g., tet) is utilizedto drive the expression of the polypeptide, protein or enzyme factor.

Preferred gene delivery vectors suitable for use within the presentinvention may be generated from retroviruses such as FIV or HIV.

Utilizing the methods and gene delivery vectors provided herein a widevariety of retinal diseases may be readily treated or prevented,including for example, macular degeneration, diabetic retinopathies,inherited retinal degeneration such as retinitis pigmentosa, glaucoma,retinal detachment or injury and retinopathies. Genes encoding a widevariety of polypeptides, proteins or enzymes may be employed, includingthose which, when expressed, prevent or alleviate the effects of thelysosomal storage disorder. An example is β-glucuronidase.

The invention therefore relates to a method of treating or preventingretinal diseases of the eye, comprising, administering intravitreously agene delivery vector which directs the expression of a polypeptide,protein or enzyme, such that said retinal disease of the eye is treatedor prevented.

In certain embodiments, the protein, polypeptide or enzyme is selectedfrom the group consisting of β-glucuronidase; neuraminidase;sphingomyelinase; sulfatases; arylsulfatase β; β-galactosidase;α-galactosidase; ceramidase; glucocerebrosidase; β-hexosaminidase;galactosylceramidase; arylsulfatase A; α-N-acetylgalactosaminidase;aspartylglycosaminidase; α-L-fucosidase; α-mannosidase; β-mannosidase;sialidase; iduronate sulfatase; α-L-iduronidase; GalNac-4-sulfatase; Gal6-sulfatase; heparin N-sulfatase; α-N-acetylglucosaminidase; acetyl-CoA;GInNAc 6-sulfatase; α-glucosidase; acid lipase;6-phospho-N-acetylglycosamine transferase; α-neuraminidase;gangliosidase; tripeptidyl protease; CLN3; and palmitoyl proteinthioesterase (PPT).

The invention further relates to treating retinal disease of the eyesuch as macular degeneration; diabetic retinopathy, inherited retinaldegeneration, such as retinitis pigmentosa; and glaucoma.

According to the invention, the said gene delivery vector is aretrovirus selected from the group consisting of HIV and FIV.

The invention further provides methods of treating diseases includingSly syndrome; Salla disease; infantile sialic acid storage disease;cystinosis; Morbus Gaucher disease; type 1 sialidosis; Batten's disease;Mucolipidosis Type IV; Hernansky-Pudlak syndrome; gangliosidosis;galactosialidosis; Type B Niemann-Pick disease; multiple sulfatasedeficiency; Austin's disease; Morquio syndrome; arylsulfatase Bdeficiency; neuraminidase deficiency; β-galactosidase deficiency;Hurler's disease; Hunter's disease; Fabry disease; Farber disease;metachromatic leukodystropy; Niemann-Pick disease; Schindler disease;aspartylglycosaminuria; fucosidosis; α-mannosidosis; β-mannosidosis;sialidosis; Maroteaux-Lamy syndrome; Sanfilippo syndrome; Pompe disease(glycogenosis II); Wolman disease; I-cell disease; pseudo Hurlerpolydystrophy; and Krabbe disease.

According to a preferred embodiment, the invention provides a method oftreating or preventing cell damage in retinal epithelial cellsassociated with Sly syndrome in a human comprising administering to thehuman a gene delivery vector that directs the expression ofβ-glucuronidase.

In a particularly preferred embodiment, the gene delivery vector is FIV.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a comparison of liver sections from a normal (1A)and MPS VII-affected (1B) mouse.

FIGS. 2A and 2B show a comparison of brain sections from a normal (2A)and MPS VII-affected (2B) mouse.

FIGS. 3A–3C show the result of gene transfer to the eye afterintravitreous injection of FIVβgal with evidence of gene transfer to theiris (3A) and retina (3B and 3C, a higher power view).

FIG. 4 shows that gene transfer with an FIV vector expressing atherapeutic gene product allows for prolonged expression ofβ-glucuronidase and extensive activity of β-glucuronidase throughout thebrain.

FIG. 5 shows correction of the pathologic defect in MPS VII mice when agene is expressed from this FIV vector. There is correction in theregion where the virus was injected (5B) and at remote sites (5C). 5A isa control tissue from a normal mouse.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a polypeptide” includes a mixture of two or morepolypeptides, and the like.

DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Gene delivery vehicle” refers to a construct which is capable ofdelivering, and, within preferred embodiments expressing, one or moregene(s) or sequence(s) of interest in a host cell. Representativeexamples of such vehicles include viral vectors, nucleic acid expressionvectors, naked DNA, and certain eukaryotic cells (e.g., producer cells).

The terms “lentiviral vector construct,” “lentiviral vector,” and“recombinant lentiviral vector” are used interchangeably herein andrefer to a nucleic acid construct derived from a lentivirus whichcarries, and within certain embodiments, is capable of directing theexpression of a nucleic acid molecule of interest. Lentiviral vectorscan have one or more of the lentiviral wild-type genes deleted in wholeor part, as described further below, but retain functional flankinglong-terminal repeat (LTR) sequences (also described below). FunctionalLTR sequences are necessary for the rescue, replication and packaging ofthe lentiviral virion. Thus, a lentiviral vector is defined herein toinclude at least those sequences required in cis for replication andpackaging (e.g., functional LTRs) of the virus. The LTRs need not be thewild-type nucleotide sequences, and may be altered, e.g., by theinsertion, deletion or substitution of nucleotides, so long as thesequences provide for functional rescue, replication and packaging.

Generally, a lentiviral vector includes at least one transcriptionalpromoter or promoter/enhancer or locus defining element(s), or otherelements that control gene expression by other means such as alternatesplicing, RNA export, post-translational modification of messenger, orpost-transcriptional modification of protein. As explained above, suchvector constructs also include a packaging signal, LTRs or functionalportions thereof, and positive and negative strand primer binding sitesappropriate to the retrovirus used (if these are not already present inthe retroviral vector). Optionally, the recombinant lentiviral vectormay also include a signal that directs polyadenylation, selectableand/or non-selectable markers, an origin of second strand DNA synthesis,as well as one or more restriction sites and a translation terminationsequence. Examples of markers include, but are not limited to, neomycin(Neo), thymidine kinase (TK), hygromycin, phleomycin, puromycin,histidinol, green fluorescent protein (GFP), human placental alkalinephosphatase (PLAP), DHFR, β-galactosidase and human growth hormone(hGH). By way of example, such vectors typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second strand DNAsynthesis, and a 3′ LTR or a portion thereof.

The terms “FIV retroviral vector construct,” “FIV vector,” and“recombinant FIV vector” are used interchangeably to refer to alentiviral vector construct, as defined above, which includes one ormore FIV sequences. By way of example, such vectors typically include a5′ FIV LTR, a primer binding site, a packaging signal, an origin ofsecond strand DNA synthesis, and a 3′ FIV LTR. Heterologous sequencesthat are included in the vector construct are those which encode aprotein, such as an enzyme, the expression of which is deficient in theselected target cells.

“Expression cassette” refers to an assembly which is capable ofdirecting the expression of the sequence(s) or gene(s) of interest. Theexpression cassette includes a promoter or promoter/enhancer which isoperably linked to (so as to direct transcription of) the sequence(s) orgene(s) of interest, and often includes a polyadenylation sequence aswell. Within certain embodiments of the invention, the expressioncassette described herein may be contained within a plasmid construct.In addition to the components of the expression cassette, the plasmidconstruct may also include a bacterial origin of replication, one ormore selectable markers, a signal which allows the plasmid construct toexist as single-stranded DNA (e.g., a M13 origin of replication), atleast one multiple cloning site, and a “mammalian” origin of replication(e.g., a SV40 or adenovirus origin of replication).

“Packaging cell” refers to a cell which contains those elementsnecessary for production of infectious recombinant retrovirus which arelacking in a recombinant retroviral vector. Packaging cells contain oneor more expression cassettes which are capable of expressing proteinswhich encode gag, pol and env-derived proteins. Packaging cells can alsocontain expression cassettes encoding one or more of vif, rev, or ORF 2in addition to gag/pol and env expression cassettes.

“Producer cell” and “Vector Producing Cell Line” (VCL) refer to a cellwhich contains all elements necessary for production of recombinantvector particles.

“Lentiviral vector particle” as used herein refers to a recombinantlentivirus which carries at least one gene or nucleotide sequence ofinterest, which is generally flanked by lentiviral LTRs. The lentivirusmay also contain a selectable marker. The recombinant lentivirus iscapable of reverse transcribing its genetic material into DNA andincorporating this genetic material into a host cell's DNA uponinfection. Lentiviral vector particles may have a lentiviral envelope, anon-lentiviral envelope (e.g., an amphotropic or VSV-G envelope), achimeric envelope or a modified envelope (e.g., truncated envelopes orenvelopes containing hybrid sequences).

“FIV vector particle” as utilized herein refers to a lentiviralparticle, as defined above, which is derived from FIV.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. Gene13:197, 1981. Such techniques can be used to introduce one or moreexogenous DNA moieties, such as a plasmid vector and other nucleic acidmolecules, into suitable host cells. The term refers to both stable andtransient uptake of the genetic material.

The term “transduction” denotes the delivery of a DNA molecule to arecipient cell either in vivo or in vitro, via a replication-defectiveviral vector, such as via a recombinant lentiviral vector particle.

The term “heterologous” as it relates to nucleic acid sequences such asgene sequences and control sequences, denotes sequences that are notnormally joined together, and/or are not normally associated with aparticular cell. Thus, a “heterologous” region of a nucleic acidconstruct or a vector is a segment of nucleic acid within or attached toanother nucleic acid molecule that is not found in association with theother molecule in nature. For example, a heterologous region of anucleic acid construct could include a coding sequence flanked bysequences not found in association with the coding sequence in nature.Another example of a heterologous coding sequence is a construct wherethe coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, acell transformed with a construct which is not normally present in thecell would be considered heterologous for purposes of this invention.Allelic variation or naturally occurring mutational events do not giverise to heterologous DNA, as used herein.

The term “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription and translation of a coding sequence in arecipient cell. Not all of these control elements need always be presentso long as the selected coding sequence is capable of being replicated,transcribed and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct-the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

For the purpose of describing the relative position of nucleotidesequences in a particular nucleic acid molecule throughout the instantapplication, such as when a particular nucleotide sequence is describedas being situated “upstream,” “downstream,” “5,” or “3” relative toanother sequence, it is to be understood that it is the position of thesequences in the non-transcribed strand of a DNA molecule that is beingreferred to as is conventional in the art.

By “isolated” when referring to a nucleotide sequence, is meant that theindicated molecule is present in the substantial absence of otherbiological macromolecules of the same type. Thus, an “isolated nucleicacid molecule which encodes a particular polypeptide” refers to anucleic acid molecule which is substantially free of other nucleic acidmolecules that do not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50%, preferably at least about 75%, more preferably at leastabout 80%–85%, preferably at least about 90%, and most preferably atleast about 95%–98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying-the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353–358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482–489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which-form-stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

By “vertebrate subject” is meant any member of the subphylum chordata,including, without limitation, mammals such as cattle, sheep, pigs,goats, horses, and human and non-human primates; domestic animals suchas dogs and cats; laboratory animals including rodents such as mice,rats and guinea pigs, and the like; birds, including domestic, wild andgame birds such as cocks and hens including chickens, turkeys and othergallinaceous birds; and fish. The term does not denote a particular age.Thus, both adult and newborn animals, as well as fetuses, are intendedto be covered.

MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of compositions and methods similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

As noted above, the present invention provides compositions and methodsfor treating, preventing, or, inhibiting retinal diseases of the eye,comprising the general step of administering intravitreously arecombinant FIV vector which directs the expression of one or morepolypeptides, proteins or enzymes, such that the retinal disease of theeye is treated or prevented. The invention also provides compositionsand methods for treating, preventing, or inhibiting diseases of thebrain related to lysosomal storage disorders. In order to further anunderstanding of the invention, a more detailed discussion is providedbelow regarding (A) gene delivery vectors; (B) polypeptides, proteins orenzymes for use in treating lysosomal storage diseases; and (C) methodsof administering the gene delivery vectors in the treatment orprevention of retinal diseases of the eye and diseases of the brain.

A. Gene Delivery Vectors

1. Construction of Retroviral Gene Delivery Vectors

Within one aspect of the present invention, retroviral gene deliveryvehicles are provided which are constructed to carry or express aselected gene(s) or sequence(s) of interest. Briefly, retroviral genedelivery vehicles of the present invention may be readily constructedfrom a wide variety of retroviruses, including for example, B, C, and Dtype retroviruses as well as spumaviruses and lentiviruses such as FIV,HIV, HIV-1, HIV-2 and SIV (see RNA Tumor Viruses, Second Edition, ColdSpring Harbor Laboratory, 1985). Such retroviruses may be readilyobtained from depositories or collections such as the American TypeCulture Collection (“ATCC”; 10801 University Blvd., Manassas, Va.20110–2209), or isolated from known sources using commonly availabletechniques.

Any of the above retroviruses may be readily utilized in order toassemble or construct retroviral gene delivery vehicles given thedisclosure provided herein, and standard recombinant techniques (e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, 1989; Kunkle, PNAS 82:488, 1985). Inaddition, within certain embodiments of the invention, portions of theretroviral gene delivery vehicles may be derived from differentretroviruses. For example, within one embodiment of the invention,retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNAbinding site from a Rous Sarcoma Virus, a packaging signal from a MurineLeukemia Virus, and an origin of second strand synthesis from an AvianLeukosis Virus.

Within one aspect of the present invention, retrovector constructs areprovided comprising a 5′ LTR, a tRNA binding site, a packaging signal,one or more heterologous sequences, an origin of second strand DNAsynthesis and a 3′ LTR, wherein the vector construct lacks gag/pol orenv coding sequences.

Within certain embodiments of the invention, retrovirus vectors areprovided wherein viral promoters, preferably CMV or SV40 promotersand/or enhancers are utilized to drive expression of one or more genesof interest.

Within other aspects of the invention, retrovirus vectors are providedwherein tissue-specific promoters are utilized to drive expression ofone or more genes of interest.

Retrovirus vector constructs for use with the subject invention may begenerated such that more than one gene of interest is expressed. Thismay be accomplished through the use of di- or oligo-cistronic cassettes(e.g., where the coding regions are separated by 120 nucleotides orless, see generally Levin et al., Gene 108:167–174, 1991), or throughthe use of Internal Ribosome Entry Sites (“IRES”).

Within one aspect of the invention, self-inactivating (SIN) vectors aremade by deleting promoter and enhancer elements in the U3 region of the3′ LTR, including the TATA box and binding sites for one or moretranscription factors. The deletion is transferred to the 5′ LTR afterreverse transcription and integration in transduced cells. This resultsin the transcriptional inactivation of the LTR in the provirus. Possibleadvantages of SIN vectors include increased safety of the gene deliverysystem as well as the potential to reduce promoter interference betweenthe LTR and the internal promoter which may result in increasedexpression of the gene of interest. Furthermore, it is reasonable toexpect tighter control of regulatable gene therapy vectors due to thelack of an upstream promoter element in the 5′ LTR.

FIV vectors are particularly preferred for use herein. FIV vectors maybe readily constructed from a wide variety of FIV strains.Representative examples of FIV strains and molecular clones of suchisolates include the Petaluma strain and its molecular clones FIV34TF10and FIV14 (Olmsted et al., PNAS 86:8088–8092, 1989; Olmsted et al., PNAS86:2448–2452, 1989; Talbot et al., PNAS 86:5743–5747, 1989), the SanDiego strain and its molecular clone PPR (Phillips et al., J. Virology64:4605–4613, 1990), the Japanese strains and their molecular clonesFTM191CG and FIV-TM2 (Miyazawa et al., J. Virology 65:1572–1577, 1991)and the Amsterdam strain and its molecular clone 19K1 (Siebelinket al.,J. Virology 66:1091–1097–1992). Such FIV strains may either be obtainedfrom feline isolates, or more preferably, from depositories orcollections such as the ATCC, or isolated from known sources usingcommonly available techniques.

Any of the above FIV strains may be readily utilized in order toassemble or construct FIV gene delivery vehicles given the disclosureprovided herein, and standard recombinant techniques (e.g., Sambrook etal., Molecular Cloning: A laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, 1989; Kunkle, PNAS 82:488, 1985; InternationalPublication Nos. WO 99/15641 and WO 99/36511). In addition, withincertain embodiments of the invention, portions of the FIV gene deliveryvehicles may be derived from different viruses. For example, within oneembodiment of the invention, recombinant FIV vector or LTR sequences maybe partially derived or obtained from HIV, a packaging signal from SIV,and an origin of second strand synthesis from HIV-2.

Within one aspect of the present invention, FIV vector constructs areprovided comprising a 5′ FIV LTR, a tRNA binding site, a packagingsignal, one or more heterologous sequences, an origin of second strandDNA synthesis, an RNA export element and a 3′ FIV LTR. Briefly, LongTerminal Repeats (“LTRs”) are subdivided into three elements, designatedU5, R and U3. These elements contain a variety of signals which areresponsible for the biological activity of a retrovirus, including forexample, promoter and enhancer elements which are located within U3.LTRs may be readily identified in the provirus (integrated DNA form) dueto their precise duplication at either end of the genome. For purposesof the present invention, a 5′ FIV LTR should be understood to includeas much of the native 5′ FIV LTR in order to function as a 5′ promoteror promoter/enhancer element to allow reverse transcription andintegration of the DNA form of the vector. The 3′ FIV LTR should beunderstood to include as much of the 3′ FIV LTR to function as apolyadenylation signal to allow reverse transcription and integration ofthe DNA form of the vector.

Additionally, FIV vector constructs may contain hybrid FIV LTRs where upto 75% of the wildtype FIV LTR sequence is deleted and replaced by oneor more viral or non-viral promoter or promoter/enhancer elements (e.g.,other retroviral LTRs and/or non-retroviral promoters orpromoter/enhancers such as the CMV promoter/enhancer or the SV40promoter) similar to the hybrid LTRs described by Chang, et al., J.Virology 67, 743–752, 1993; Finer, et al., Blood 83, 43–50, 1994 andRobinson, et al., Gene Therapy 2, 269–278, 1995.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, tRNA binds to aretroviral tRNA binding site by Watson-Crick base pairing; and iscarried with the retrovirus genome into a viral particle. The tRNA isthen utilized as a primer for DNA synthesis by reverse transcriptase.The tRNA binding site may be readily identified based upon its locationjust downstream from the 5′ LTR. Similarly, the origin of second strandDNA synthesis is, as its name implies, important for the second strandDNA synthesis of a retrovirus. This region, which is also referred to asthe poly-purine tract, is located just upstream of the 3′ LTR.

The packaging signal sequence of FIV directs packaging of viral geneticmaterial into the viral particle. A major part of the packaging signalin FIV lies between the 5′ FIV LTR and the gag/pol sequence with thepackaging signal likely overlapping in part with the 5′ area of thegag/pol sequence.

In addition to 5′ and 3′ FIV LTRs, a tRNA binding site, a packagingsignal, and an origin of second strand DNA synthesis, certain preferredrecombinant FIV vector constructs for use herein also comprise one ormore genes of interest, each of which is discussed in more detail below.In addition, the FIV vectors may, but need not, include an RNA exportelement (also variously referred to as RNA transport, nuclear transportor nuclear export elements) which may be the FIV RRE (Rev-responsiveelement) or a heterologous transport element. Representative examples ofsuitable heterologous RNA export elements include the Mason-Pfizermonkey virus constitutive transport element, the MPMV CTE (Bray et al.,PNAS USA 91, 1256–1260, 1994), the Hepatitis B Virus posttranscriptionalregulatory element, the HBV PRE (Huang et al., Mol. Cell. Biol.13:7476–7486, 1993 and Huang et al., J. Virology 68:3193–3199, 1994),other lentiviral Rev-responsive elements (Daly et al., Nature342:816–819, 1989 and Zapp et al., Nature 342:714–716, 1989) or the PREelement from the woodchuck hepatitis virus. Further RNA export elementsinclude the element in Rous sarcoma virus (Ogert et al., J. Virology70:3834–3843, 1996; Liu & Mertz, Genes & Dev. 9:1766–1789, 1995) and theelement in the genome of simian retrovirus type 1 (Zolotukhin et al., J.Virology 68:7944–7952, 1994). Other potential elements include theelements in the histone gene (Kedes, Annu. Rev. Biochem. 48:837–870,1970), the a interferon gene (Nagata et al., Nature 287:401–408, 1980),the β-adrenergic receptor gene (Koilka et al., Nature 329:75–79, 1987),and the c-Jun gene (Hattorie et al., PNAS 85:9148–9152, 1988).

FIV vector constructs which lack both gag/pol and env coding sequencesmay be used with the present invention. As utilized herein, the phrase“lacks gag/pol or env coding sequences” should be understood to meanthat the FIV vector contains less than 20, preferably less than 15, morepreferably less than 10, and most preferably less than 8 consecutivenucleotides which are found in gag/pol or env genes, and in particular,within gag/pol or env expression cassettes that are used to constructpackaging cell lines for the FIV vector construct. This aspect of theinvention provides for FIV vectors having a low probability ofundesirable recombination with gag/pol or env sequences which may occurin a host cell or be introduced therein, for example, by transformationwith an expression cassette. The production of FIV vector constructslacking gag/pol or env sequences may be accomplished by partiallyeliminating the packaging signal and/or the use of a modified orheterologous packaging signal. Within other embodiments of theinvention, FIV vector constructs are provided wherein a portion of thepackaging signal that may extend into, or overlap with, the FIV gag/polsequence is modified (e.g., deleted, truncated or bases exchanged).Within other aspects of the invention, FIV vector constructs areprovided which include the packaging signal that may extend beyond thestart of the gag/pol gene. Within certain embodiments, the packagingsignal that may extend beyond the start of the gag/pol gene is modifiedin order to contain one, two or more stop codons within the gag/polreading frame. Most preferably, one of the stop codons eliminates thegag/pol start site. In other embodiments, the introduced mutation maycause a frame shift in the gag/pol coding region.

Other retroviral gene delivery vehicles may likewise be utilized withinthe context of the present invention, including for example thosedescribed in EP 0,415,731; WO 90/07936; WO 91/0285, WO 9403622; WO9325698; WO 9325234; U.S. Pat. No. 5,219,740; WO 9311230; WO 9310218;Vile and Hart, Cancer Res. 53:3860–3864, 1993; Vile and Hart, CancerRes. 53:962–967, 1993; Ram et al., Cancer Res. 53:83–88, 1993; Takamiyaet al., J. Neurosci. Res. 33:493–503, 1992; Baba et al., J. Neurosurg.79:729–735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242and WO91/02805).

Packaging cell lines suitable for use with the above describedretrovector constructs may be readily prepared (see, e.g., U.S. Pat.Nos. 5,591,624 and 6,013,517, incorporated herein by reference in theirentireties; and International Publication No. WO 95/30763), and utilizedto create producer cell lines (also termed vector cell lines or “VCLs”)for the production of recombinant vector particles. Briefly, the parentcell line from which the packaging cell line is derived can be selectedfrom a wide variety of mammalian cell lines, including for example,human cells, monkey cells, feline cells, dog cells, mouse cells, and thelike.

For example, potential packaging cell line candidates are screened byisolating the human placental alkaline phosphatase (PLAP) gene from theN2-derived retroviral vector pBAAP, and inserting the gene into the FWvector construct. To generate infectious virus, the construct isco-transfected with a VSV-G encoding expression cassette (e.g., pMLP-Gas described by Emi et al., J. Virology 65, 1202–1207, 1991; or pCMV-G,see U.S. Pat. No. 5,670,354) into 293T cells, and the virus harvested 48hours after transfection. The resulting virus can be utilized to infectcandidate host cells which are subsequently FACS-analyzed usingantibodies specific for PLAP. Candidate host cells include, e.g., humancells such as HeLa (ATCC CCL 2.1), HT-1080 (ATCC CCL 121), 293 (ATCC CRL1573), Jurkat (ATCC TIB 153), supT1 (NIH AIDS Research and Referencereagent program catalog #100), and CEM (ATCC CCL 119) or feline cellssuch as CrFK (ATCC CCL 94), G355–5 (Ellen et al., Virology 187:165–177,1992), MYA-1 (Dahl et al., J. Virology 61:1602–1608, 1987) or 3201-B(Ellen et al., Virology 187:165–177, 1992). Production of p24 andreverse transcriptase can also be analyzed in the assessment of suitablepackaging cell lines.

After selection of a suitable host cell for the generation of apackaging cell line, one or more expression cassettes are introducedinto the cell line in order to complement or supply in trans componentsof the vector which have been deleted (see, e.g., U.S. Pat. Nos.5,591,624 and 6,013,517, incorporated herein by reference in theirentireties; and International Publication No. WO 95/30763). For example,packaging expression cassettes may encode either gag/pol sequencesalone, gag/pol sequences and one or more of vif, rev or ORF 2, or one ormore of vif, rev or ORF 2 alone and may contain an RNA export element.For example, the packaging cell line may contain only ORF 2, vif, or revalone, ORF 2 and vif, ORF 2 and rev, vif and rev or all three of ORF 2,vif and rev.

Packaging cell lines may also comprise a promoter and a sequenceencoding ORF 2, vif, rev, or an envelope (e.g., VSV-G), wherein thepromoter is operably linked to the sequence encoding ORF 2, vif, rev, orthe envelope. For packaging cell lines containing inducible gag/pol orenv expression cassettes, additional expression cassettes facilitatingthe transactivation of the inducible promoter may be incorporated.

The expression cassette may or may not be stably integrated. Thepackaging cell line, upon introduction of an FIV vector, may produceparticles at a concentration of greater than 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, or, 10⁹ cfu/ml.

B. Treatment of Lysosomal Storage Diseases

In humans, there are numerous inherited metabolic diseases affecting theCNS, many of which are the result of a deficiency in a soluble lysosomalenzyme, which would benefit from FIV-based gene therapy as disclosedherein. The long-lasting expression conferred by FIV-based vectors couldbe improved further if coupled to recent advancements made intranscriptional regulation of transgene expression. Such vectors mayfind application not only to correction of lysosomal storage disease inhumans, but also to diseases which may benefit from the protectiveeffects of secreted growth factors, such as Parkinson's disease,Alzheimers disease, and the dominant neurodegenerative diseases such asHuntington's and the spinal cerebellar ataxias.

Lysosomal diseases in humans that are amenable to treatment using themethods of the invention include Batten's disease (neuronal ceroidlipofuscinosis), which has autosomal recessive inheritance (Marshman etal., Aust. N. Z. J. Ophthalmol. (1998) 26:251–254); mucolipidosis typeIV, characterized by retinal degeneration and brain abnormalities (Freiet al. (1998) Neurology 51:565–569; Siegel et al. (1998) Elec. Clin.Neurophys. 106:400–403); and infantile cystinosis (Broyer, M. (1997)Rev. Prat. 47:1550–1553).

Hermansky-Pudlak syndrome, which is an often fatal autosomal recessivedisorder (Feng et al. (1997) Hum. Mol. Genet. 6:793–797), is alsosuitable for treatment according to the invention. Feng et al. describea mouse disease that is homologous to the human syndrome and provides ananimal model for the human disease, as well as for the Chediak-Higashisyndrome. Both human syndromes are characterized by lysosomal storagedefects, and HPS is a single-gene disorder. Oh et al. in Nat. Genet.14:300–306 (1996) describe a transmembrane protein that is defective inHPS patients due to truncation caused by a frameshift mutation.

Other suitable diseases include adult form galactosialidosis (Usui etal. (1993) Metab. Pediatr. Syst. Ophthalmol. 16:19–22); Salla disease(Mancini et al. (1992) Eur. J. Pediatr. 151:590–595); Type B.Niemann-Pick disease resulting from deficiency or decreased activity ofsphingomyelinase (Barton et al. (1992) Metab. Pediatr. Syst. Ophthalmol.15:16–20); multiple sulfatase deficiency, or Austin's disease (al Aqeelet al. (1992) J. Child Neurol. 7 Suppl. PS12–21); Morquio syndrome(systemic mucopolysaccharidosis IV A) which can exhibit inclusionsdistributed in various parts of the eye including the retinal pigmentepithelium (Iwamoto et al. (1990) Graefes Arch. Clin. Exp. Ophthalmol.228:342–349); and β-galactosidase deficiency (Andia et al. (1978) Clin.Genet. 14:16–23).

The methods of the invention also have use in the veterinary fieldincluding treatment of domestic pets and farm animals. Murnane at al.(1994) described an ovine form of GM-1 gangliosidosis, and detected CNSabnormalities and blindness in some animals (J. Vet. Intern. Med.8:221–223. Deficiencies of β-galactosidase and α-neuraminidase have alsobeen reported in sheep (Murnane et al. (1989) Am. J. Pathol.134:263–270), with CNS and ocular involvement. Stramm et al. ((1986)Invest. Ophthamol. Vis. Sci. 27:1050–1057) reported a deficiency ofarylsufatase B in a recessively inherited feline lysosomal storagedisease, MPS VI. Tissues affected include the retinal pigment epitheliumand other regions of the eye.

C. Method for Treating and Preventing Retinal Disease, andPharmaceutical Compositions

In one aspect, the present invention provides methods which generallycomprise the step of intravitreously administering a gene deliveryvector which directs the expression of one or more proteins,polypeptides or enzymes to the retina in order to treat, prevent, orinhibit the progression of a retinal disease. In another aspect, thepresent invention provides methods for administering a gene deliveryvehicle to the brain, wherein the expression of one or morepolypeptides, proteins, or enzymes is directed. As utilized herein, theterms “treated, prevented, or, inhibited” refer to the alteration of adisease course or progress in a statistically significant manner.Determination of whether a disease course has been altered may bereadily assessed in a variety of model systems, discussed in more detailbelow, which analyze the ability of a gene delivery vector to delay,prevent or rescue photoreceptors, as well as other retinal cells, fromcell death, or to delay or prevent cell damage or death in the brain.

1. Retinal Diseases of the Eye

A wide variety of retinal diseases may be treated given the teachingsprovided herein. For example, within one embodiment of the inventiongene delivery vectors are administered to a patient intravitreously inorder to treat or prevent macular degeneration. Briefly, the leadingcause of visual loss in the elderly is macular degeneration (MD), whichhas an increasingly important social and economic impact in the UnitedStates. As the size of the elderly population increases in this country,age related macular degeneration (AMD) will become a more prevalentcause of blindness than both diabetic retinopathy and glaucoma combined.Although laser treatment has been shown to reduce the risk of extensivemacular scarring from the “wet” or neovascular form of the disease,there are currently no effective treatments for the vast majority ofpatients with MD.

Within other embodiments, gene delivery vectors can be administered to apatient intravitreously in order to treat or prevent diabeticretinopathy, or other vascular diseases of the retina.

Within another embodiment, gene delivery vectors can be administered toa patient intravitreously in order to treat or prevent an inheritedretinal degeneration. One of the most common inherited retinaldegenerations is retinitis pigmentosa (RP), which results in thedestruction of photoreceptor cells, and the RPE. Other inheritedconditions include Sly syndrome; Bardet-Biedl syndrome (autosomalrecessive); Congenital amaurosis (autosomal recessive); Cone or cone-roddystrophy (autosomal dominant and X-linked forms); Congenital stationarynight blindness (autosomal dominant, autosomal recessive and X-linkedforms); Macular degeneration (autosomal dominant and autosomal recessiveforms); Optic atrophy, autosonial dominant and X-linked forms);Retinitis pigmentosa (autosomal dominant, autosomal recessive andX-linked forms); Syndromic or systemic retinopathy (autosomal dominant,autosomal recessive and X-linked forms); and Usher syndrome (autosomalrecessive). This group of debilitating conditions affects approximately100,000 people in the United States alone.

Within other embodiments of the invention, gene delivery vectors can beadministered to a patient intravitreously in order to treat or preventglaucoma. Glaucoma is a heterogeneous group of disorders that share adistinct type of optic nerve damage that leads to loss of visualfunction. The disease is manifest as a progressive optic neuropathythat, if left untreated, leads to blindness. It is estimated that asmany as 3 million Americans have glaucoma and, of these, as many as120,000 are blind as a result. Furthermore, it is the number one causeof blindness in African-Americans. Its most prevalent form, primaryopen-angle glaucoma, can be insidious. This form usually begins inmidlife and progresses slowly but relentlessly. If detected early,disease progression can frequently be arrested or slowed with medicaland surgical treatment.

2. Lysosomal Storage Diseases of the Brain

In the brain, both neurons and cells of glial lineage are affected. Thebrain lysosomal storage and decreased neuronal numbers may contribute tothe behavioral, memory, and cognitive deficits seem in MPS VII mice(Chang et al., Neuro Report 4:507–510, 1993). In humans, clinicalfeatures related to brain lysosomal storage include deafness and mentalretardation. The morphological aspects of hearing loss in affected miceinclude thickening of the tympanic membrane, otitis media with expansionof the middle ear mucos, deformation of the middle ear ossicles, andinner ear alterations (Berry et al., Lab. Invest. 77:438–445, 1994).

3. Methods of Administration

Gene delivery vectors are delivered to the eye by intravitreousinjection. The vitreous is approached either through the ora serata ordirectly through the pupil, negotiating the needle around the lens. Inone application, the primary target cells to be transduced are theretinal ganglion cells, which are the retinal cells primarily affectedin glaucoma. In this application, the injection volume of the genedelivery vector can large, as the volume is not constrained by theanatomy of the interphotoreceptor or subretinal space. Acceptabledosages in this instance can range from 25 μl to 1000 μl. In anotherapplication, the retinal pigment epithelium (RPE) cells are the targetcells. Both cell types are targeted by the FIV virus.

Gene delivery vectors are delivered to the brain of mice by injectioninto the straitum or right lateral ventricle. Intraventricular injectionresults in transduction of both ependyma and choroidal epithelium.

4. Assays

A wide variety of assays may be utilized in order to determineappropriate dosages for administration, or to assess the ability of agene delivery vector to treat or prevent a particular disease. Certainof these assays are discussed in more detail below.

a. Electroretinographic Analysis

Electroretinographic analysis can be utilized to assess the effect ofgene delivery administration into the retina. Briefly, animals are darkadapted overnight and then in dim red light, then anesthetized withintramuscular injections of xylazine (13 mg/kg) and ketamine (87 mg/kg).Full-field scotopic ERGs are elicited with 10-μsec flashes of whitelight and responses were recorded using a UTAS-E 2000 VisualElectrodiagnostic System (LKC Technologies, Inc., Gaithersburg, Md.).The corneas of the rats are anesthetized with a drop of 0.5%proparacaine hydrochloride, and the pupils dilated with 1% atropine and2.5% phenylephrine hydrochloride. Small contact lenses with gold wireloops are placed on both corneas with a drop of 2.5% methylcellulose tomaintain corneal hydration. A silver wire reference electrode is placedsubcutaneously between the eyes and a ground electrode is placedsubcutaneously in the hind leg. Stimuli are presented at intensities of−1.1, 0.9 and 1.9 log cd m⁻² at 10-second, 30-second and 1-minuteintervals, respectively. Responses are amplified at a gain of 4,000,filtered between 0.3 to 500 Hz and digitized at a rate of 2,000 Hz on 2channels. Three responses are averaged at each intensity. The a-wavesare measured from the baseline to the peak in the cornea-negativedirection, and b-waves are measured from the cornea-negative peak to themajor cornea-positive peak. For quantitative comparison of differencesbetween the two eyes of rats, the values from all the stimulusintensities are averaged for a given animal.

b. Retinal Tissue Analysis

Retinal tissue analysis can also be utilized to assess the effect ofgene delivery administration into the retina. This procedure isdescribed in more detail below in Example 2.

c. Neurological Function

In mice, neurological function can be measured by EEG. Behavioral,memory, and cognitive function can be assayed as described. (Chang etal., Neuro Report 4:507–510, 1993.)

d. Neural Tissue Analysis

Tissues can be harvested from treated mice or primates, and processedfor evaluation of lysosomal distension using routine procedures. In thisinvention it is useful to evaluate, for example, the ipsilateralstriatum, ipsilateral cortex, and contra-lateral cortex. Measurementsperformed over time can indicate increasing correction of cells distantto the vector administration site. CSF can also be collected andevaluated for protein levels or enzyme activity, particularly if thevector encodes a secretable enzyme.

5. Pharmaceutical Compositions

Gene delivery vectors may be prepared as a pharmaceutical productsuitable for direct administration. Within preferred embodiments, thevector should be admixed with a pharmaceutically acceptable carrier forintravitreous administration. Examples of suitable carriers are salineor phosphate buffered saline.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Restriction and modifying enzymes, as well as other reagents for DNAmanipulations were purchased from commercial sources, and used accordingto the manufacturers' directions. In the cloning of DNA fragments,except where noted, all DNA manipulations were done according tostandard procedures. See, e.g., Sambrook et al., supra.

EXAMPLE 1 β-Glucuronidase expression in Neuronal tissue of MPS VII Mice

β-glucuronidase deficiency, or MPS VII, is representative of a group oflysosomal storage disorders in humans characterized by accumulations ofproteoglycans. The disease is progressive, with most tissues affectedincluding the CNS. Mouse models for β-glucuronidase deficiency reflectcharacteristics of the human disorder, and cells in diseased tissuescontain numerous, distended lysosomes. In the brain, both neurons andcells of glial lineage are affected. FIG. 1 illustrates the VII mice(1B) compared to normal liver (1A). FIG. 2 illustrates lysosomalgranules in brain of MPS VII mice (2B) compared to normal brain (2A).

A. FIV vectors expressing β-glucuronidase were generated which weredevoid of vif and ORF 2 (FIVβglucΔvifΔorf2), as described below inExample 1B and disclosed in International Publication No. WO 99/36511,published Jul. 22, 1999. Vectors were injected into the striatum ofβ-glucuronidase deficient mice, and animals sacrificed 3 to 18 weekslater and tissues analyzed for transgene expression, enzyme activity,and correction of pathology.

FIVβgluc vector-mediated gene transfer resulted in robust levels ofexpression in the injected hemisphere, 3 weeks after injection ofvector. A representative coronal section from a mouse sacrificed 3 weeksafter injection is shown in FIG. 4. The level of activity did notdecline by 18 weeks, and there was no evidence of inflammatoryinfiltrate.

In situ RNA analysis for human β-glucuronidase mRNA confirmed thattransduction was limited to cells near the injection site, and suggeststhat mRNA and/or virus was not transported to distant cells. Thus, theextensive distribution of enzyme likely results from secretion bytransduced cells, followed by distribution via the CSF and extracellularfluids, with uptake by distant cells.

Tissues harvested from β-glucuronidase deficient mice injected into theright striatum with FIVβgluc vector were also processed for evaluationof lysosomal distension. In tissues from mice sacrificed 3 weeks aftergene transfer, correction was noted in the ipsilateral striatum,ipsilateral cortex, and modest reductions in storage product were notedin the contralateral cortex. By 6 weeks, there was dramatic restorationof cellular morphology in both hemispheres of the brain (FIG. 5). Thisextensive correction was maintained at 18 weeks the last time pointstudied because of the shortened life spans of these animals(approximately 6 months). The results suggest that persistent expressionof this enzyme, approximately 2–5% of which is secreted, can result incorrection of cells at increasing distances over time; This resultindicates that focal transduction with a gene transfer vector expressinga soluble lysosomal enzyme can lead, eventually, to wide-spreaddissemination of the enzyme. This is enhanced by stable expression byFIV-based vectors devoid of ORF 2 and vif.

β-glucuronidase deficient mice show progressive impairment in neurologicfunction as measured by EEG. In β-glucuronidase deficient mice givenFIVβgluc vector, the progressive impairment is attenuated, presumably byexpression of functional enzyme and clearance of storage product.

These results indicate that FIV-based vectors, devoid of accessoryproteins, can globally correct a severe neurologic deficit in the brainsof mice suffering from lysosomal storage disease.

B. The effects of FIVβglucΔvifΔorf2 gene transfer on learning and memorywas also evaluated after injection into 15 wk oldβ-glucuronidase-deficient mice with established cellular deficits. FIVvectors for use in this example were generated essentially as describedin International Publication No. WO 99/36511, published Jul. 22, 1999.Specifically, FIV packaging constructs were generated in a series ofsteps from the full-length FIV molecular clone, FIV-34TF10 (NIH AIDSResearch and Reference Reagent Program, Cat. No. 1236; Phillips et al.,J. Virol. 66: 5464, 1992, Talbott et al., PNAS 86: 5743, 1989) asdescribed (Johnston et al., J Virol 73:4991–5000, 1999). The FIV vectorconstruct, pVET_(L)Cβgal (pVET_(L)Cβ in ref (Johnston et al., J Virol73:4991–5000, 1999), was generated by inserting an expression cassetteconsisting of the CMV promoter followed by the β-galactosidase gene intothe PVET_(L) FIV vector backbone. The PVET_(L) backbone contains the FIV5′ LTR, in which the FIV U3 region is replaced by the CMVpromoter/enhancer, 0.5 kb of Gag coding region, a multicloning site andthe FIV 3′ LTR (Johnston et al., J Virol 73:4991–5000, 1999). Toconstruct pVET_(L)Rβgluc, an RSV promoter lacking a functionalpolyadenylation signal was first liberated from pUC19RSV by digestionwith BamHI and SalI. The resulting fragment was inserted into similarlydigested PVET_(L) to generate PVET_(L)RSV. Next, the β-glucuronidasecDNA was liberated from pAdRSV4 by digestion with Xho I and theresulting fragment ligated into Sal I digested/CIP treated PVET_(L)RSVto generate pVET_(L)Rgluc (+polyA). A portion of the β-glucuronidasecDNA was amplified by PCR and the product digested with Bgl II and Xho Iin order to remove the polyadenylation signal from the cDNA. To generatepVET_(L)Rgluc, the resulting fragment was joined in a three-way ligationwith an Nco I/Bgl II fragment and a Xho I/Nco I fragment frompVET_(L)Rgluc (+polyA). All constructs were screened by restrictionenzyme digestion and the sequence of regions amplified by PCR confirmedby sequence analysis. Oligonucleotides were synthesized by OperonTechnologies, Inc. (Alameda, Calif.) and sequences as well as moredetailed cloning methods are available upon request. Construction of theVSV-G envelope expression plasmid, pCMV-G, has been described (Yee et.al., PNAS 91:9564, 1994). Pseudotyped FIVβgluc and FIVβgal vectorparticles were generated by transient transfection of plasmid DNA into293T cells plated one day prior to transfection at a density of 2.8×10⁶cells per 10 cm diameter culture dish. Cotransfections were performedusing a 1:2:1 molar ratio of FIV packaging construct, FIV vectorconstruct and VSV-G envelope-expressing plasmid. DNA complexes wereprepared using calcium phosphate (Profectin kit; Promega Corp. Madison,Wis.) and transfected into cells according to the manufacturer'sinstructions. The medium was replaced 8–16 hr after transfection and thesupernatant harvested twice between 32 and 48 hr after the start oftransfection. The harvested supernatants were filtered through a 0.45 MNalgene filter and stored at −70° C. or concentrated prior to storage.Supernatants were concentrated by centrifugation (Johnston et al., JVirol 73:4991–5000). Vector titers were determined on HT1080 cells byserial dilution and assay for β-galactosidase or β-glucuronidaseexpression (Li et al., PNAS 92:7700–7704, 1995).

C57BL/6 mice were obtained from Harlan Sprague (Indianapolis, Ind.).These mice are deficient in β-glucuronidase and provide an acceptedanimal model for the study of lysosomal storage disease. Mice with adeficiency in this soluble lysosomal enzyme manifest both visceral andCNS manifestations, thus recapitulating the human syndrome, also knownas Sly syndrome or MPS VII. The inability to appropriately degradeproteoglycans leads to progressive accumulation of precursor products inthe lysosomes. In this storage disease enzyme production and release bya subpopulation of parenchymal cells within the brain results inwidespread clearing of metabolic precursors. For virus injections, theβ-glucuronidase-deficient mice were anesthetized with ketamine/xylazine(ketamine 100–125 mg/kg, xylazine 10–12.5 mg/Kg). The bregma was thenexposed by incision and used as a zero coordinate and injections madestereotactically into the striatum or ventricle as previously described(Ghodri et al., Hum Gene Ther 9:2331–2340, 1998).

For histological studies the mice were injected unilaterally with 5 μl(intraparenchymal) or 10 μl (intraventricular) of FIVβgal, FIVβgalΔvif,FIVβgalΔorf2, or FIVβgalΔvifΔorf2 or FIVβglucΔvifΔorf2. Animals weresacrificed at 3, 6, 9, 15, and 18 weeks after gene transfer and brainsanalyzed for enzyme activity, volume analysis, in-situ RNAhybridization, and analysis of storage vacuoles as previously described(Li et al., PNAS 92:7700–7704, 1995).

Delivery of FIVβglucΔvifΔorf2 to the brains of eight-week oldβ-glucuronidase deficient mice resulted in transduction of cells nearthe injection site. However, the level of enzyme activity measured bythe histological assay extended well beyond the focus of transducedcells, with 20–25% of the hemisphere positive for enzyme activity, andwas relatively stable. Tissues harvested from β-glucuronidase-deficientmice injected into one hemisphere of the brain with FIVβglucΔvifΔorf2were examined for the effects of gene therapy on lysosomal distension.In tissues from mice sacrificed 3 weeks after gene transfer,histological correction was observed in the ipsilateral striatum,ipsilateral cortex, and modest reductions in storage product seen in thecontralateral cortex. No inflammation was found in brains of animalssacrificed at this time. By 6 weeks, there was dramatic restoration ofcellular morphology in both hemispheres of the brain indicative ofcross-correction. The absence of lysosomal inclusions was maintainedthrough the course of the study (18 weeks) suggesting that persistentexpression of this enzyme from transduced cells, approximately 2–5% ofwhich may be secreted, results in correction of cells at increasingdistances over time.

These findings have direct therapeutic implications because they showthat focal transduction with lentivirus-based vectors expressing asoluble lysosomal enzyme can lead to wide-spread dissemination of thegene product.

Gene therapy-mediated correction of the histopathological defects in theCNS of animals with established, advanced, neurodegenerative disease hasnot been shown to result in improved neurological function. Such animprovement after gene therapy would indicate that the CNS disease mightin part be reversible. Accordingly, the ability of FIVβglucΔvifΔorf2 torestore or improve CNS function was evaluated. Specifically, the affectof FIV-mediated gene transfer of FIVβglucΔvifΔorf2 on spatial learningability was-examined. The repeated acquisition and performance chamber(RAPC) was utilized to assess spatial learning and memory in both gus+/− and gus −/− mice (Brooks et al., Repeated Acquisition andPerformance Chamber for Mice: A Paradigm for Assessment of SpatialLearning and Memory. Neurobiology of Learning & Memory, (In Press)). Inbrief, mice were first deprived of water for 12–16 hours and habituatedto a saccharin solution before being introduced into the RAPC for thefirst time. A 0.2% solution of saccharin dissolved in water was providedfor 30 minutes twice a day for 2 days, after which regular drinkingwater was provided ad libitum. Subsequently, all mice were given fourapparatus habituation sessions, each allowed to freely explore thechambers and consume saccharin drops: 1) Placed in front and in back ofall doors with all doors taped open (session A); 2) in front and in backof all doors with all doors unlatched (session B); 3) only in back of Cand D doors with all doors unlatched (session C); 4) only in back of Ddoors with all doors unlatched (session D).

Following habituation, mice were tested over the course of fourexperimental sessions (sessions ½ and ¾ were separated by 5 weeks). A 12hour water deprivation period preceded all behavioral test sessions,which occurred approximately every third day, with ad libitum water onnon-test days for the remainder of the study. Each session consisted ofthree presentations each of the repeated acquisition (RA) component andthe performance (P) component. In the RA component, the specific doorsequence changed unpredictably with each successive test session (nottrial) according to a matrix that prevented the same door on a givenpanel from being open on consecutive sessions. During the P component,the sequence of doors leading to saccharin was constant across sessions.A static audio signal was played for the duration of the P component asa discriminative stimulus signaling-that component, whereas the absenceof the audio signal served as the discriminative stimulus for the RAcomponent. A total of three trials (from goal box to saccharin) werecarried out during each presentation of the RA and P components during asession, for a total of 18 trials per session. Latency was measured asthe time required for a subject to leave the start box, successfullynavigate through the four compartments, and consume the saccharinsolution in the goal box. Mice were manually placed in the goal box inthe event of failure to reach it within 10 minutes on any trial. Inaddition to latency, the number and sequence of door errors made by thesubject were also recorded.

Initial RAPC experiments were performed on untreatedβ-glucuronidase-deficient and age-matched, heterozygous, control animalsto define baseline learning and performance abilities. At 8 weeks of agecomparison of the number of errors made by the β-glucuronidase deficientand control mice indicated that the β-glucuronidase deficient mice havea baseline impairment in learning. Baseline differences (pre-treatment)in numbers of errors and latencies in the RAPC were evaluated usingrepeated measures analyses of variance with component (learning andperformance) and session (1–4) as within group factors andβ-glucuronidase status (+/− vs −/−) as a between-group factor. Thesewere followed, where appropriate by one factor ANOVAs (β-glucuronidasestatus) for individual session data. Statistical assessment of changesin these measures post-treatment were carried out separately for eachtreatment (FIVβgal and FIVβgluc) and for each component (learning andperformance) in RMANOVAs with β-glucuronidase status (+/− vs −/−) as abetween-group factor and session (10–14) as within group factors.Subsequent one factor ANOVAs were used where appropriate for determiningdifferences between +/− vs −/− groups for each session. Fourteen daysfollowing gene transfer animals were re-assessed in the RAPC asdescribed above. Three sessions were conducted post-operatively (eachsession separated by one week) to assess the effects of FIV mediatedgene transfer.

Baseline Differences between +/− vs. −/− Groups: β-glucuronidasedeficient mice (−/−) showed significantly greater numbers of errors inthe RAPC in both the learning and the-performance component of thesession over the 4 baseline sessions in which they were tested (maineffect of group: F(1,12)=742.05, p=0.0001) with subsequent post-hocassessments confirming differences between the two groups during eachsession and in both the learning and performance components (all pvalues <0.05). Mean group error values of the +/− group in the learningcomponent ranged from 32 to 47 whereas corresponding values for the −/−mice were 66 to 88. Similarly, numbers of errors in the performancecomponent of the +/− group ranged from 10 to 16 with values of the grouphigher at 24 to 29. Latencies and error number in the RAPC learningcomponent increased in β-glucuronidase deficient mice tested between the8th and 13th week of age indicating progressive impairment of cognitivefunction.

Latency values between the +/− and −/− groups: Latency values alsodiffered between the +/− and −/− groups (main effect of group:F(1,12)=6.17, p=0.029). In the learning component, −/− mice actuallyexhibited significantly shorter latencies during the first session, butthese subsequently increased, such that by sessions 3 and 4, latenciesof the −/− group were significantly higher than those of the +/− group(approximately 165 vs. 120 sec.). Similarly, latency values of the −/−group were also significantly shorter during the first 2 sessions in theperformance component of the schedule (approximately 25 vs. 50 sec.) butrose over the subsequent time period such that by session 4 theirlatency values significantly exceeded those of the +/− group (5.1 vs. 59sec) and this represented almost a doubling of latencies across time forthe −/− group.

Post-Treatment Differences: Mice from the −/− group that receivedFIVβgal continued to show significantly greater numbers of errors inboth the learning and the performance components relative to +/− micecorrespondingly treated (F(1,3)=386.4, p=0.0003 and F(1,3)=262.98,p=0.0005) for the learning and performance components, respectively).The magnitude of the differences remained comparable to those seen priorto treatment. Similarly, −/− mice that received FIVβgal also continuedto sustain longer latencies than did +/− mice (F(1,3)=107.5, p=0.0019and F(1,3)=12.96, p=0.037, respectively, for the learning andperformance components). These differences were sustained acrosssessions as indicated by the absence of any significant interaction ofgroup by sessions in these analyses.

In contrast, mice from the −/− group that received FIVβgluc no longerevidenced any differences from the +/− mice that received FIVβgluc inthe number of errors in the learning component (main effect of group,both p values >0.05). Numbers of errors for the −/− and +/− groupsaveraged approximately 36 and 39, respectively in the learningcomponent. Correspondingly, the numbers of errors of −/− mice thatreceived FIVβgluc were initially higher than those of the +/− mice thatreceived FIVβgluc, but they declined across the course of sessions(interaction of group by session=F(1,6)=28.3, p=0.0009) such that by thefinal session, values no longer differed (p=0.18). Similarly, latencydifferences that were observed between the +/− and −/− groups prior totreatment were no longer evident following FIVβgluc in either thelearning (p=0.18) or the performance component (p=0.32) of the schedule.

RAPC tests done 2 and 3 weeks after gene transfer demonstrated markeddifferences between the experimental groups. The data from the RAPCtests acquired after gene transfer indicated a significant improvementin the learning component in β-glucuronidase deficient mice thatreceived FIVβglucΔvifΔorf2. Interestingly, there was no statisticaldifference between β-glucuronidase deficient mice injected withFIVβglucΔvifΔorf2 and FIVβgalΔvifΔorf2-injected control mice. Becausethere was no decline between the FIVβgalΔvifΔorf2 heterozygous controlmice from their preinjections values, the βgal gene transfer itself didnot reduce the performance or learning components of the control group.Thus, FIVβglucΔvifΔorf2 gene transfer had a profound positive impact onthe progressive neurodegenerative disease in the β-glucuronidasedeficient mouse model. The RAPC data indicate that gene transferrestored cognitive function to brains of β-glucuronidase deficient mice.

The combined results are important because they show that FIV-basedvectors, devoid of accessory proteins, can reverse a severe neurologicdeficit in the brains of mice with an established lysosomal storagedisease.

EXAMPLE 2 Transgenic RAT S334TER as a Model for PhotoreceptorDegeneration

This example describes the S334ter transgenic rat as a model forphotoreceptor degeneration. Briefly, rhodopsin is a seven-transmembraneprotein found in photoreceptor outer segments, which acts as aphotopigment. The S334ter mutation results in the truncation of theC-terminal 15 amino acid residues of rhodopsin and is similar torhodopsin mutations found in a subset of patients with retinitispigmentosa (RP). RP is a heterogeneous group of inherited retinaldisorders in which individuals experience varying rates of vision lossdue to photoreceptor degeneration. In many RP patients, photoreceptorcell death progresses to blindness. Transgenic S334ter rats are bornwith normal number of photoreceptors. The mutant rhodopsin gene beginsexpression at postnatal day 5 in the rat, and photoreceptor cell deathbegins at postnatal day 10–15. In transgenic line S334ter-3,approximately 70% of the outer nuclear layer has degenerated by day 60in the absence of any therapeutic intervention. The retinal degenerationin this model is consistent from animal to animal and follows apredictable and reproducible rate. This provides an assay fortherapeutic effect by morphological examination of the thickness of thephotoreceptor nuclear layer and comparison of the treated eye to theuntreated (contralateral) eye in the same individual animal.

A. Retinal Tissue Analysis

The rats are euthanized by overdose of carbon dioxide inhalation andimmediately perfused intracardially with a mixture of mixed aldehydes(2% formaldehyde and 2.5% glutaraldehyde). Eyes are removed and embeddedin epoxy resin, and 1 μm thick histological sections are made along thevertical meridian. Tissue sections are aligned so that the ROS andMuller cell processes crossing the inner plexiform layer are continuousthroughout the plane of section to assure that the sections are notoblique, and the thickness of the ONL and lengths of RIS and ROS aremeasured. These retinal thickness measurements are plotted and establishthe baseline retinal degeneration rates for the animal model. Theassessment of retinal thickness is as follows: briefly, 54 measurementsof each layer or structure are made at set points around the entireretinal section. These data are either averaged to provide a singlevalue for the retina, or plotted as a distribution of thickness orlength across the retina.

For FIV vector evaluation experiments in vivo, a suitable line oftransgenic rats is TgN(s334ter) line 4 (abbreviated s334ter 4).Expression of the mutated opsin transgene begins at postnatal day P5 inthese rats, leading to a gradual death of photoreceptor cells. Theserats develop an anatomically normal retina up to P15, with the exceptionof a slightly increased number of pyknotic photoreceptor nuclei in theouter nuclear layer (ONL) than in non-transgenic control rats. In thisanimal model, the rate of photoreceptor cell death is approximatelylinear until P60, resulting in loss of 40–60% of the photoreceptors.After P60, the rate of cell loss decreases, until by one year theretinas have less than a single row of photoreceptor nuclei remaining.

EXAMPLE 3 β-Glucuronidase or β-Galactosidase Expression AfterFIV-Mediated Gene Transfer in Retinal Pigment Epithelium ofβ-Glucuronidase-Deficient Mice

A. In this example, the cellular targets for transduction followingintravitreal (corneal and ora serata) injection of FIVβgal (felineimmunodeficiency virus expressing E. coli β-galactosidase) were tested.One microliter was injected intravitreally. Results show that bothcorneal endothelium and cells of the iris could be transduced.Intravitreal injection of FIBβgal also resulted in very efficienttransduction of the RPE.

Immunohistochemistry following intravitreal injection of a mixture ofconfirmed AdGFP (adenovirus expressing green fluorescent protein) andFIVβgal confirmed that both viruses could mediate transduction ofcorneal endothelium and cells of the iris (FIG. 3A), and that FIV couldalso transduce cells in the retina (FIGS. 3B and 3C). In some cases,photoreceptor cells were also transduced following intravitrealinjection of FIVβgal. Transgene expression with FIVβgal remainedrelatively stable for 21 days, the last time point tested. The efficacyof intravitreal injection of FIVβgluc (FIV expressing β-glucuronidase)was tested using an animal model of RPE-dependent photoreceptor celldegeneration, the β-glucuronidase deficient mouse. Intravitrealinjection of FIVβgluc to the eyes of β-glucuronidase deficient miceresulted in rapid reduction (within 2 weeks) of the lysosomal storagedefect within the RPE.

Vector is administered to the vitreous as described in Li, T. AndDavidson, B. L. (1995) Proc. Natl. Acad. Sci. 92:7700–7704.β-glucuronidase expression is measured as described by Li and Davidson(1995).

B. β-glucuronidase-deficient and Balb/C mice were used to assess theability of FIV vector particles to transfer genes intravitreally. Balb/Cmice were used for β-gal and eGFP injections due to the lack ofpigmentation in the eye, therefore allowing the transgene product to bevisualized easier. FIV packaging constructs were generated in severalsteps from the full-length FIV molecular clone, FIV-34TF10 (NIH AIDSResearch and Reference Reagent Program, Cat. No. 1236; Phillips et al.,J. Virol. 66: 5464, 1992, Talbott et al., PNAS 86: 5743, 1989) asdescribed above in Example 1B. In particular, the FIV vector constructswere generated by insertion of an expression cassette into the PVET_(L)FIV vector backbone (Johnston et al., J Virol 73:4991–5000, 1999).β-galactosidase expression was driven by the CMV promoter, while theβ-glucuronidase expression was driven by the RSV promoter (Johnston etal., J Virol 73:4991–5000, 1999). The construction of VSV-g envelopeexpression plasmid, pCMV-G, has been previously described (Yee et al.,PNAS 91:9564–9568, 1994). The generation of psuedotyped FIVβgal andFIVβgluc vector particles through transient transfection has beendescribed (Johnston et al., J Virol 73:4991–5000, 1999).

All animals used in this example were between 4 and 8 weeks old andweighed between 12 and 24 grams. Mice were anesthetized withKetamine-Xylazine (ketamine, 100–125 mg/kg; xylazine, 10–12.5 mg/kg).Eyes were dilated with 0.2% cyclopentolate, 0.5% phenylephrine, and0.05% tropicamide. After dilation a drop of 0.5% proparacaine wasadministered as a topical anesthetic. A microscalpel was used to make asmall self closing incision in the cornea just central to the freeborder of the iris. A 5 microliter Hamilton syringe with a fixed 1 inchblunt 33 gauge needle (Reno, Nev.) was inserted through the incision inthe cornea and slid between the iris and the lens into the posteriorchamber of the eye where 1–2 microliters of virus (FIV, 1×10⁸ TU, Ad1×10¹⁰ IU) or saline was injected into the vitreous. Injections wereobserved at low magnification with a stereo microscope. The incision wascoated with antibiotic ointment to help prevent leakage and infection.To aid recovery, the animals were injected with 1 milliliter normalsaline subcutaneously and placed under a heat lamp.

Animals sacrificed for histochemical analysis were anesthetized andperfused with 2% paraformaldehyde in PBS. Eyes were enucleated andpostfixed for 4 hours in 2% paraformaldehyde, after postfixation thelens was removed and the eyes were cryoprotected in 30% sucrose in PBS.The eyes were frozen in O.C.T. (Sakura Finetek U.S.A., Torrence, Calif.)and 10-μm slide sections were prepared.

For β-galactosidase staining, slides were rinsed with PBS and thenreacted with 35 mM K₃Fe(CN)₆, 35 mM K₄Fe(CN)₆, 2 mM MgCl₂, and5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal, 1 mg/ml; Sigma,St. Louis, Mo.) in PBS for 4 hours at 37° C. to identify β-galactosidaseactivity.

To visualize β-glucuronidase activity, slides were fixed with anacetone-formalin solution, washed two times for 5 minutes with 0.05 Msodium acetate buffer, pH 4.5, and incubated in 0.25 mMNaph-As-Bi-β-glucuronidide (Sigma, St. Louis, Mo.) in 0.05 M sodiumacetate buffer, pH 4.5, all at 4° C. The slides were then developed for2 hours at 37° C. with 0.25 mM Naph-As-Bi-β-glucuronidide in 0.05 Msodium acetate buffer, pH 5.2, with 1:500 2% hexazotized pararosaliline(Sigma) (Bancroft, 1982; Roessler, 1994). Slides were subsequently driedand coverslipped with permount (Fisher).

For co-infection experiments, equal volumes of FIVβgal and AdeGFP (FIV,1×10⁸ TU, Ad 1×1¹⁰ IU) were mixed and 2 μl injected intravitreally. Tovisualize both transgenes simultaneously, we stainedimmunohistochemically with a rabbit polyclonal antibody forβ-galactosidase (Sigma, St. Louis, Mo.) followed by a rhodaminesecondary antibody (Jackson). The rabbit polyclonal antibody waspreabsorbed for 24 hours on naive mouse tissue. All antibodies werediluted in 3.0% BSA crystilline fraction (Sigma, St. Louis, Mo) and 0.3%saponin (Sigma, St. Louis, Mo.) in PBS. Fluorescence was observed with aLeica DMRBE and digital images captured with a spot rt camera.

Animals for morphological analysis were sacrificed at 2, 7, or 12 weekspost-injection. Animals were anesthetized and perfused with 2%paraformaldehyde, 2.5% gluteraldehyde in PBS. Eyes were enucleated andpostfixed in the same fixative for 4 hours. The lenses were removed andthe eyes were further postfixed in 1% OsO₄ in PBS. After fixation, eyeswere dehydrated and embedded in SPURRS resin (EMS). Blocks weresectioned with a diamond knife (EMS) at 90 m and mounted on coppergrids. The sections were stained with lead citrate and uranel acetateand viewed on a Hitachi 7000 transmission electron microscope.

As explained above, Balb/C mice were used for β-gal and eGFP injectionsdue to the lack of pigmentation in the eye, therefore allowing thetransgene product to be visualized easier. Eyes were evaluated at either1 or 3 weeks after FIVβgal injection. β-galactosidase-positive cellswere found predominantly in the retinal pigmented epithelium (RPE).Positive cells were also seen in the ciliary process, iris, and cornealendothelium. The distribution of positive cells did not vary over time.

These results were dissimilar to previously published adenoviralintravitreal injection data. An intravitreal injection of recombinantadenovirus results in mainly corneal endothelium and iris cells positivefor the transgene and no positive cells in the RPE, this result issimilar to the results published by Li et al., Invest Opthalmol Vis Sci35:2543–2549, 1994. To test whether the positive cells in the RPE weredue to the FIV vector or the injection technique a mixing experiment wasperformed. Equal volumes of Ad5RSVeGFP and FIVβgal were mixed andinjected 2.0 μl in the vitreous. GFP and gal positive cells were bothpresent in the anterior chamber of the eye, but only β-gal positivecells were found in the RPE. Interestingly, there was an increase in thenumber of β-gal positive cells found in the corneal endothelium when theFIV was injected in concert with the AV.

Treated MPS mouse eyes were examined for β-glucuronidase activity byhistochemical stain as described above. The red precipitate reactionproduct may be obscured by the pigmentation in areas of the eye. Due tothe secreted nature of β-glucuronidase positive staining cells werefound throughout the eye at all time points (2, 7, and 12 weeks). Theseeyes were analyzed for the presence of distended lysosomes usingelectron microscopy. The three main cell types that were evaluated fordistended lysosomal presence were the corneal endothelium, nonpigmentedepithelium of the ciliary process, and the retinal pigmented epithelium.Buffer injected and non-treated eyes showed numerous large distendedlysosomes within the cytoplasmic space in all of these cell types aswell as other cell types throughout the eye. In the cases of the threeaforementioned cell types, all appeared swollen and larger than thecells in age-matched control animals. In the case of the RPE the pigmentgranules were displaced to the apical surface. However, the neuralretina remained relatively unaffected with very few cells containingnotable distended lysosomes.

At two weeks post-injections of FIVβgluc, the pathological differenceswere striking under low magnification electron microscopy (2,000×). Thedistended lysosomes had near completely disappeared in all three celltypes evaluated. The phenotypic correction was most dramatic in the RPEand nonpigmented epithelium of the ciliary process as the cells of thetreated animals were almost indistinguishable from those of the controlnormal animals. The correction of the corneal endothelium was also quitedramatic but not as complete as found in the other cell types. Therewere still some small distended lysosomes found in some of the cells.These results held true for all time points tested with one surpriseaddition at the 12 week time point. In addition to the cornealendothelium being partially phenotypically corrected, the distendedlysosomes within the keratocytes of the corneal stroma were alsosignificantly reduced.

In addition to looking for phenotypical correction of the lysosomaldistention in the eye, β-glucuronidase enzyme levels were measured indifferent ocular tissues. At the time of sacrifice the fluid was removedfrom the eye and the cornea and retina were dissected out for analysis.

These results show that after an intravitreal injection of recombinantFIV encoding for the β-glucuronidase gene, there was significantamelioration of the distended lysosomal phenotype in several of theaffected eye tissues. Surprisingly, in addition to the expectedcorrection of the phenotype in the RPE and corneal endothelium as shownwith adenoviral gene therapy to the MPS VII mouse eye (Li and Davidson,PNAS 92:7700–7704, 1995) correction was observed in the non-pigmentedepithelium of the ciliary process and the keratocytes of the cornealstroma. Correction of the phenotype was not limited to the cells thatwere infected with the virus as shown by FIVβgal injections. Cornealendothelial cells and the non-pigmented epithelium of the ciliaryprocess were sporadically infected and no positive keratocytes werefound. Therefore correction of the phenotype in these cells is mostlikely attributable to the uptake of extracellular β-glucuronidase thathas been secreted by transduced cells.

EXAMPLE 4 FIV-Mediated Gene Transfer to the CNS and Liver ofβGlucuronidase Deficient Mice

An FIV vector encoding the gene for β-glucuronidase (FIVβgluc) wasinjected into the CNS (Striatum) and systemic circulation ofβ-glucuronidase deficient mice. The distribution of enzyme activity andthe extent of pathological correction were assessed. β-Glucuronidaseactivity was extensive in the ipsilateral stratum 21 days postintraparenchymal injection of FIVβgluc to the CNS. Activity was alsorobust in the ipsilateral frontal and parietal cortex, the ependymalcells lining the ventricles, and the corpus callosum. In thecontralateral hemisphere enzyme activity was noted but to a lesserdegree. Animals sacrificed at 42, 84, and 126 days following genetransfer continued to exhibit sustained levels of enzyme activity in thesame regions. Semi-thin sections from the striatum and cortex ofβ-glucuronidase deficient animals were analyzed for lysomalaccumulation. There was a marked reduction in the number of distendedvacuoles in both neurons and glial cells bilaterally, indicatingcorrection of pathology.

Twenty-one days after intravenous administration of FIVβgluc into thetail veins of deficient mice, histological sections showedβ-glucuronidase activity localized to both hepatocytes and Kupffer cellsin the liver. Quantitative measurements using a fluorometric assaydetected β-glucuronidase activity corresponding to 2% of wild-typeactivity. Analysis of semi-thin section of liver showed this amount ofactivity to be partially corrective, significantly reducing lysosomaldistention in hepatocytes. These data indicate that FIV-mediated genetherapy is effective in an in vivo model for transduction of all celltypes in the CNS and liver. Furthermore, reversal of the pathology canbe achieved and maintained in both organ systems.

Accordingly, lentiviral vectors and methods of using the same for thetreatment of brain and eye lysosomal storage disorders have beendisclosed. From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the appended claims.

1. A method of providing β-glucuronidase to retinal epithelial cells ofa vertebrate subject, said method comprising administering to thesubject intravitreally a lentiviral vector particle wherein saidlentiviral vector particle comprises a lentiviral vector, comprising a5′ lentiviral long terminal repeat (LTR), a tRNA binding site, apackaging signal, a promoter operably linked to a polynucleotideencoding β-glucuronidase, an origin of second strand DNA synthesis and a3′ lentiviral LTR, under conditions whereby the β-glucuronidase encodedby said lentiviral vector is expressed in retinal epithelial cells ofsaid vertebrate subject.
 2. The method of claim 1, wherein saidlentiviral vector comprises one or more LTRs selected from the groupconsisting of a 5′ LTR and a 3′ LTR from a virus selected from the groupconsisting of human immunodeficiency virus (HIV), human immunodeficiencyvirus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), felineimmunodeficiency virus (FIV), and simian immunodeficiency virus (SIV).3. The method of claim 2, wherein said lentiviral vector comprises oneor more LTRs selected from the group consisting of a 5′ LTR and a 3′ LTRfrom FIV.
 4. A method of treating cell damage in retinal epithelialcells of a vertebrate subject, wherein the cell damage is associatedwith Sly syndrome, said method comprising administering to the subjectintravitreally feline immunodeficiency virus (FIV) vector particlewherein said FIV vector particle comprises an FIV vector comprising a 5′FIV long terminal repeat (LTR), a tRNA binding site, a packaging signal,a polynucleotide encoding β-glucuronidase operably linked to an FIV LTRpromoter or a promoter element, an origin of second strand DNA synthesisand a 3′ FIV LTR, under conditions whereby the β-glucuronidase encodedby said FIV vector is expressed at a level sufficient to treat damage inretinal epithelial cells of said vertebrate subject.
 5. The method ofclaim 1 or 4, wherein the promoter is a CMV, RSV, or SV40 promoter.